MicroRNAs (miRNA) are a class of multifunctional small (18-25 nucleotides) noncoding RNA molecules. To date, approximately 940 miRNAs have been described. Their functions include epigenetic control of gene expression, mRNA degradation, and suppression of mRNA translation. These diverse functions of miRNAs are necessary for normal development, metabolism, cellular differentiation, proliferation, cell-cycle control, and cell death. Aberrant miRNA expression and/or activity have been implicated in a variety of human diseases including cancer.
Several studies have analyzed miRNA expression pattern in primary tumors of various types, and specific subtypes of cancers could easily be differentiated based on the expression pattern of these miRNAs. Recent studies have identified miRNAs in extracellular space, mainly through a ceramide-dependent secretory exosomes or microvesicles. These secreted miRNAs are functional and enter heterotypic cells to alter migration/invasive properties. However, secretion or packaging of miRNAs into the exosomes is a selective process as the level of miRNA in exosomes secreted by a cell type does not always correlate with the intracellular levels of the corresponding miRNA. Specific cellular proteins, most of them are RNA binding proteins, are suggested to be involved in exosomal secretion of miRNAs and their stability in circulation.
There are several reports describing differential blood/plasma/serum miRNA levels between healthy people and those with various diseases including cancer. Serum miRNA was first reported in diffuse large B-cell lymphoma; sera of patients contained higher levels of miR-155, miR-210, and miR-21. Elevated serum miR-21 levels correlated with good prognosis. Similar studies in prostate cancer revealed elevated levels of miR-141 in the plasma of cancer patients compared to healthy patients, although the same result was not obtained in another study. A four-miRNA predictive profile from serum has been described recently for non-small-cell lung cancer. There are limited studies on breast cancer. One study reported higher serum levels of miR-155 in the progesterone receptor (PR)-positive breast cancer patients compared with PR-negative breast cancer patients. Two recent studies reported elevated levels of miR-195 and let-7a in the whole blood of breast cancer patients; levels of these miRNAs declined after surgical removal of tumors suggesting that they were tumor derived. Elevated levels of miR-195 in the whole blood appear to be unique to breast cancer. Elevated levels of plasma miR-122 and miR-192 were reported after acetaminophen-induced liver injury suggesting that tissues that are enriched for specific miRNAs may release them upon injury.
It is postulated that the miRNAs are released into circulation either actively by the tumor cells or passively as a result of tumor cell death and lysis. However, this does not explain low serum levels of some miRNAs in cancer patients compared with healthy controls. For example, plasma of patients with acute myeloid leukemia show low levels of miR-92a compared with healthy despite of high levels of this miRNA in leukemic cells. In the sera of lung cancer patients, 28 miRNAs are missing and 63 new miRNA species are detectable compared with healthy. Similarly, sera of ovarian cancer patients show elevated levels of five miRNAs and decreased levels of three miRNAs compared with healthy.
These observations raise questions as to whether serum miRNAs in cancer patients are directly derived from tumor cells or an indirect consequence of effects of cancer on other tissues, which then release miRNA into circulation. Considering that the tumor often represents very tiny portion of the body mass, microvesicles/exosomes secreted from the tumor cells are less likely to be sufficient enough to change miRNA profile in a large volume of blood (five liters in a 72-kg person). Systemic effects of cancer on distant organs could easily result in differential serum miRNA profile in cancer patients. More importantly, these changes in serum profile could persist even after the patient is “disease free” if an epigenetic mechanism is involved in the systemic effects. In the latter situation, miRNAs would be poor markers of active disease.
To address the above issues, we determined the levels of breast cancer-associated miRNAs in the sera of healthy and breast cancer patients who are considered clinically cancer-free at the time of serum collection. Further validation of significant initial results were performed with an independent sample set comprising serum from healthy, clinically disease-free breast cancer patients, and patients with overt metastasis and an additional set with serum from healthy and active metastasis patients. We report that SNORD44, a small nucleolar RNA (also called RNU44) is similar in the sera of healthy and clinically cancer-free breast cancer patients in both sets of experiments. However, levels of U6 (also called RNU6-1), which is commonly used for the purpose of normalization between samples, and U6:SNORD44 ratio were elevated in the sera of breast cancer patients, who did not have active disease. Elevated U6 was detected in the sera of both estrogen receptor alpha positive (ER+) and ER-negative breast cancer patients. Sera of patients with overt metastasis also showed elevated U6 or U6:SNORD44 ratio when compared with healthy women. Taken together, these results suggest that elevated U6 serum levels represent persistent systemic effects of breast cancer attained during cancer progression.